It is often desirable to maintain well-defined concentrations of a given compound in the blood stream for a long time. This would for instance be the case when an immunogen is administered and a strong immune response is desired, or when a peripheral therapeutic target has to be exposed continuously to a therapeutic agent for a long time. Currently there are no universally applicable strategies to enhance the intravascular half-life of any type of compound.
The number of known endogenous peptides and proteins with interesting biological activities is growing rapidly, also as a result of the ongoing exploration of the human genome. Due to their biological activities, many of these peptides and proteins could in principle be used as therapeutic agents. Endogenous peptides are, however, not always suitable as drug candidates because these peptides often have half-lives of few minutes due to rapid degradation by peptidases and/or due to renal filtration and excretion in the urine. It has been shown by others (Zobel et al., Bioorg. Med. Chem. Lett. 2003, 13, 1513-1515) that the plasma half-life of a given peptide may be significantly enhanced by reversible attachment of this peptide to plasma proteins, such as albumin or gamma-globulin. Human serum albumin (HSA) has, for instance, a half-life of more than one week. This reversible attachment requires a compound (=affinity tag) which can be linked to the therapeutic agent, and which has a high binding affinity to albumin while bound to said therapeutic agent. Thus, generally applicable affinity tags would be of great general interest, in particular as potential drug-delivery systems.
Many of the most potent albumin-ligands known are carboxylic acids, such as fatty acids, arylacetic acids (e.g. ketoprofen), or iophenoxate, or dicarboxylic acids such as 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) (Kratochwil et al. Biochemical Pharmacology 2002, 64, 1355-1374):

All the structural features present in these compounds are, however, responsible for binding, and the affinity to HSA is lowered when these structural features are modified chemically. Thus, esters or amides of the acids mentioned above will have significantly lower affinities to HSA. The reason for this is that the carboxylic acid functionality forms strong ionic bonds with basic amino acids present in the plasma protein, and is therefore responsible to a large extent for the binding to the protein. Accordingly, it is not immediately obvious how to connect one of the known, strongly HSA-binding carboxylic acids to a therapeutic agent without loosing the required affinity to HSA. A method for achieving this would, however, be highly desirable, because many, structurally diverse carboxylic acids which bind to plasma proteins are already known, and no tedious screening for new compounds would be required. Furthermore, many of the known, HSA-binding carboxylic acids are currently marketed drugs, and their metabolites have been shown to be pharmaceutically and toxicologically acceptable.
The present invention intends to provide a versatile delivery system for therapeutic agents, such as proteins, peptides, or small molecule drugs, by covalently binding these therapeutic agents to a plasma-protein ligand, capable of reversible binding to one or several plasma proteins. We have designed a novel linking strategy which enables the covalent binding of known, plasma-protein binding carboxylic acids to a broad variety of therapeutic peptides or proteins, or to any other type of compound, of which a prolonged peripheral exposure at well-defined concentrations is required. This strategy consists in converting said plasma-protein binding carboxylic acid into a carboxylic acid mimetic, having a pKa between −5 and +7, in order to be ionized to a significant extent in plasma. Such a carboxylic acid mimetic could be, for instance, an N-acylsulfonamide, which contains an additional functional group which enables covalent binding of the acid mimetic to a therapeutic agent. This linking strategy should not significantly disturb the structure of the plasma-protein binding acid, if a suitable acid mimetic has been chosen. For instance, N-acylsulfonamides are similarly acidic as carboxylic acids, and will be deprotonated and negatively charged at physiological pH (B. J. Bakes, J. A. Ellman. J. Am. Chem. Soc. 1994, 116, 11171-11172).

Other carboxylic acid mimetics may, however, also be suitable for the purpose presented above. These include, for instance, imides, N-acylureas, and N-acylcarbamates.

Alternatively, acylated derivatives of electron-deficient, amino-substituted heterocycles or arenes may also be sufficiently acidic to be deprotonated in plasma, and are therefore also comprised within the scope of this invention.

As a further alternative, carboxylic acids with a high affinity to a plasma protein may be used to C-acylate a cyano acetic acid derivative, a 3-ketocarboxylic acid derivative, or another acetic acid derivative with an electron-withdrawing group M attached to position 2. This group M could be, for example hydrogen, fluorine, —CN, —CO-alkyl, —CO-aryl, —CO2-alkyl, —NO2, —SO2-aryl, —SO2-alkyl, and the like. The resulting products would be strongly C—H-acidic and highly enolized, and could therefore also serve as carboxylic acid mimetic.

As illustrated by the sketches above, there may be an optional spacer X and a linker Y between the acid mimetic and the therapeutic agent for covalent attachment of the affinity tag to the therapeutic agent. These two groups will establish the distance between the plasma-protein binding fragment and the therapeutic agent. The linker Y can result from any reactive functional group Y1 able to form a covalent, metabolically stable bond to the therapeutic agent which will not be cleaved at significant rates in vivo. Reactive groups Y1 include, but are not limited to, carboxylic acids, amines, thiols, isothiocyanates, isocyanates, chloroformiates, O-succinimidyl carbonates, epoxides, sulfonyl chlorides, alkyl halides, electron-deficient alkenes, or other, related functional groups. Alternatively, the linker Y may also contain a metabolically labile bond, what would lead to a slow release of the untagged therapeutic agent in vivo. Such metabolically labile bonds are, for instance, present in certain carboxylic acid amides, disulfides, carboxylic esters, or carbamates.
Depending on the precise therapeutic target and on the length of the spacer X it may happen that the tagged therapeutic agent will exert its activity while bound to a plasma protein, or it could also happen that the tagged therapeutic agent will show a diminished biological activity while bound to the plasma protein, and only the unbound fraction of tagged therapeutic agent display the full biological activity. All these different features are included within the scope of the present invention.